This invention relates to methods for binding a protein with an antibody reagent such as is performed in an immunoassay. More particularly, the invention relates to methods for denaturing a disulfide-crosslinked protein to enhance the ability of an antibody directed thereto to bind with the protein.
Immunoassays are methods for the detection or determination of substances of analytical interest (analytes) based on the binding of such substances by antibody reagents. Current technology permits the development of antibodies against a wide variety of analytes. Immunoassays are particularly useful in diagnostic medicine where analytes of clinical importance are often present at low concentrations in the presence of many structurally similar background substances.
It is now known that the antigenicity of proteins, that is, the ability of proteins to be bound by antibodies directed against the protein, in certain circumstances can critically require, or be increased by, subjecting the protein to denaturing conditions prior to contact with antibody. In certain circumstances, a desired epitope on a protein can be sterically hindered, occluded, or buried in the native three-dimensional folded structure of the protein. Denaturation can relieve the steric constraints on the epitope, thereby rendering it available or more available for antibody binding. Thus, denaturation can be significant in the development of an immunoassay where available antibody reagents do not bind with the native form of the protein or where binding is too weak for the concentration of protein to be detected.
For example, U.S. Pat. No. 4,658,022 describes a general principle that the binding of antibodies directed to linear peptide epitopes in proteins can be obtained or enhanced by denaturation of the protein. This has been particularly applied in the case of proteins in which the epitope that characterizes it relative to other proteins that are present in a test sample is sterically hindered in the native form of the protein. An example is the glycated form of hemoglobin known as hemoglobin A1c. The patent describes a wide variety of means for denaturing a protein for the purposes of creating or enhancing antigenicity, including heating, sonication, treatment at high or low pH, and treatment with chemical denaturants and chaotropic agents such as guanidine, urea, and detergents. It is further reported that inclusion of reagents such as mercaptoethanol or dithiothreitol which reduce disulfide bonds can be effective promoters of the denaturation process.
It is known that the structure of many proteins includes disulfide linkages between adjacent peptide chains. Such linkages are important in defining the native three-dimensional structure of such proteins. Studies of protein structure often involve the cleavage of disulfide linkages. Such cleavage is required for complete denaturation of the protein and is generally accomplished by exposure to reducing agents (e.g., mercaptoethanol or dithiothreitol) followed by the addition of capping agents (e.g., iodoacetic acid or iodoacetamide).